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Article

A Systematic Approach to Assess the Activity and Classification of PCSK9 Variants

1
Department of Molecular Biophysics, Biofisika Institute, University of Basque Country and Consejo Superior de Investigaciones Científicas (UPV/EHU, CSIC), 48940 Leioa, Spain
2
Center for Cooperative Research in Biomaterials (CIC biomaGUNE), Basque Research and Technology Alliance (BRTA), 20014 Donostia San Sebastian, Spain
3
Inserm, UMR 1188 Diabète Athérothrombose Thérapies Réunion Océan Indien (DéTROI), Université de La Réunion, 97400 Saint-Denis de La Reunion, France
4
Fundación Biofisika Bizkaia, 48940 Leioa, Spain
5
Department of Biochemistry and Molecular Biology, Universidad del País Vasco UPV/EHU, 48080 Bilbao, Spain
*
Authors to whom correspondence should be addressed.
Kepa B. Uribe and Kevin Chemello contributed equally to this work.
Academic Editor: Łukasz Bułdak
Int. J. Mol. Sci. 2021, 22(24), 13602; https://doi.org/10.3390/ijms222413602
Received: 28 September 2021 / Revised: 3 December 2021 / Accepted: 16 December 2021 / Published: 18 December 2021
Background: Gain of function (GOF) mutations of PCSK9 cause autosomal dominant familial hypercholesterolemia as they reduce the abundance of LDL receptor (LDLR) more efficiently than wild-type PCSK9. In contrast, PCSK9 loss of function (LOF) variants are associated with a hypocholesterolemic phenotype. Dozens of PCSK9 variants have been reported, but most remain of unknown significance since their characterization has not been conducted. Objective: Our aim was to make the most comprehensive assessment of PCSK9 variants and to determine the simplest approach for the classification of these variants. Methods: The expression, maturation, secretion, and activity of nine well-established PCSK9 variants were assessed in transiently transfected HEK293 cells by Western blot and flow cytometry. Their extracellular activities were determined in HepG2 cells incubated with the purified recombinant PCSK9 variants. Their binding affinities toward the LDLR were determined by solid-phase immunoassay. Results: LDLR expression increased when cells were transfected with LOF variants and reduced when cells were transfected with GOF variants compared with wild-type PCSK9. Extracellular activities measurements yielded exactly similar results. GOF and LOF variants had increased, respectively reduced, affinities for the LDLR compared with wild-type PCSK9 with the exception of one GOF variant (R218S) that showed complete resistance to inactivation by furin. All variants were expressed at similar levels and underwent normal maturation and secretion patterns except for two LOF and two GOF mutants. Conclusions: We propose that transient transfections of HEK293 cells with a plasmid encoding a PCSK9 variant followed by LDLR expression assessment by flow cytometry is sufficient to reliably determine its GOF or LOF status. More refined experiments should only be used to determine the underlying mechanism(s) at hand. View Full-Text
Keywords: PCSK9; LDL; cholesterol; dyslipidaemias; lipoproteins; receptors; gain of function; loss of function; in vitro characterization; familial hypercholesterolemia PCSK9; LDL; cholesterol; dyslipidaemias; lipoproteins; receptors; gain of function; loss of function; in vitro characterization; familial hypercholesterolemia
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MDPI and ACS Style

Uribe, K.B.; Chemello, K.; Larrea-Sebal, A.; Benito-Vicente, A.; Galicia-Garcia, U.; Bourane, S.; Jaafar, A.K.; Lambert, G.; Martín, C. A Systematic Approach to Assess the Activity and Classification of PCSK9 Variants. Int. J. Mol. Sci. 2021, 22, 13602. https://doi.org/10.3390/ijms222413602

AMA Style

Uribe KB, Chemello K, Larrea-Sebal A, Benito-Vicente A, Galicia-Garcia U, Bourane S, Jaafar AK, Lambert G, Martín C. A Systematic Approach to Assess the Activity and Classification of PCSK9 Variants. International Journal of Molecular Sciences. 2021; 22(24):13602. https://doi.org/10.3390/ijms222413602

Chicago/Turabian Style

Uribe, Kepa B., Kevin Chemello, Asier Larrea-Sebal, Asier Benito-Vicente, Unai Galicia-Garcia, Steeve Bourane, Ali K. Jaafar, Gilles Lambert, and César Martín. 2021. "A Systematic Approach to Assess the Activity and Classification of PCSK9 Variants" International Journal of Molecular Sciences 22, no. 24: 13602. https://doi.org/10.3390/ijms222413602

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